Manufacturing Bits: Jan. 14

Tracking cell movement
Using a technology called cyro-electron microscopy (cryo-EM), Sanford Burnham Prebys Medical Discovery Institute (SBP) and the University of North Carolina at Chapel Hill (UNC-Chapel Hill) have gained a better understanding of how cells move in living organisms.

Cells, the basic building blocks of living things, need to move. Moving cells help enable embryonic development, boost the immune system, and speed up the healing process. But not all cell movement is healthy. “Tumors are most dangerous when cancer cells gain the ability to travel throughout the body (metastasis),” according to researchers at SBP. “Certain bacteria and viruses can harness the cells’ motility machinery to invade our bodies.”

So understanding how cells move is critical as a means to prevent disease. In the study, SBP and UNC-Chapel Hill used a cryo-electron microscope and machine learning to gain insights into how cells move. A form of transmission electron microscopy (TEM), cryo-EM is used to study a sample at cryogenic temperatures. A gas is assumed to be cryogenic if it can be liquefied at or below −150 °C.

Cyro-EM is often used in structural biology. In one application, a cryo-EM is used to freeze biomolecules mid-movement. Then, the structure is imaged at atomic resolutions. The system allows researchers to produce films that reveal how molecules interact with each other.

SBP and UNC-Chapel Hill used the technology to take images of the fibroblast of a mouse. A fibroblast is a type of cell. It produces collagen and other fibers. Then, the images are compared to light images of fluorescent Rac1, “a protein that regulates cell movement, response to force or strain (mechanosensing) and pathogen invasion,” according to researchers.

Using cyro-EM, researchers have identified “a densely packed, disorganized, scaffold-like structure comprised of short actin rods. These structures sprang into view in defined regions where Rac1 was activated,” according to researchers. In order words, researchers have visualized a structure that resembles a haystack, which was developed in response to a cellular signal. This in turn expands the understanding of how cells move.

SBP and the University of North Carolina at Chapel Hill have identified a dense, dynamic and disorganized actin filament nanoscaffold—resembling a haystack—that is induced in response to a molecular signal, pictured at right. Actin at rest is pictured at left. (Source: SBP)

“We were surprised that experiment after experiment revealed these unique hotspots of unaligned, densely packed actin rods in regions that correlated with Rac1 activation,” says Niels Volkmann, a professor in the Bioinformatics and Structural Biology Program at SBP. “We believe this disorder is actually the scaffold’s strength—it grants the flexibility and versatility to build larger, complex actin filament architectures in response to additional local spatial cues.”

Dorit Hanein, a professor in the Bioinformatics and Structural Biology Program at SBP, added: “This study is only the beginning. Now that we developed this quantitative nanoscale workflow that correlates dynamic signaling behavior with the nanoscale resolution of electron cryo-tomography, we and additional scientists can implement this powerful analytical tool not only for deciphering the inner workings of cell movement but also for elucidating the dynamics of many other macromolecular machines in an unperturbed cellular environment.

“Cyro-electron microscopy is revolutionizing our understanding of the inner workings of cells,” Hanein said. “This technology allowed us to collect robust, 3D images of regions of cells—similar to MRI, which correlates detailed images of our body. We were able to visualize cells in their natural state, which revealed a never-before-seen actin nano-architecture within the cell.”

More specifically, researchers imaged the proteins responsible for cell-cell contacts. Researchers used this technique to observe proteins, which are critical for tissues and organs.

Cryo-electron tomography is much like cryo-EM. A sample is frozen and examined with an electron microscope. Cyro-electron tomography takes images from different directions. The images are assembled into a 3D image by a computer.

With the technology, researchers published the first 3D images of human skin at molecular resolution. “This is a real breakthrough in two respects,” said Achilleas Frangakis, group leader at EMBL. “Never before has it been possible to look in three dimensions at a tissue so close to its native state at such a high resolution. We can now see details at the scale of a few millionths of a millimeter. In this way we have gained a new view on the interactions of molecules that underlie cell adhesion in tissues – a mechanism that has been disputed over decades.”

U-ExM is a form of super-resolution microscopy. With the technology, researchers took images of multi-protein complexes. The complex is anchored in a polymer. “With U-ExM, we can really depict ultrastructural details. The method is reliable,” said Markus Sauer from JMU. “And it delivers a picture that is four times higher resolved than with standard methods of microscopy.”